Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

Hiroshi Matsushita ,Mamoru Satoh,Hideaki Shimada,Shuji Kubo,Masafumi Matsuo,Fumio Nomura,Takeshi Tomonaga,Bahityar Rahmutulla,Masahiko Hatano,Chiaki Nakaseko,Satoru Miyagi,Atsushi Kaneda,Nobuko Tanaka,Atsushi Iwama,Takayuki Ishige,Sakae Itoga,Takashi Miki,Minoru Kito,Masaki Fukuyo,Tyuji Hoshino,Takeshi Mori,Kazuo Kïtamura ,Yaeko Nakajima‐Takagi

doi:10.18632/oncotarget.3244

Abstract

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Highlights

DNA damage affects carcinogenesis, transcription, alternative splicing, and cell cycle control; the precise mechanism behind these affects remains largely unexplored
Activated c-Myc accelerates the cell cycle by suppressing p27Kip1 expression, which leads to the accumulation of DNA damage [14, 15].These results suggest that disturbed FBPinteracting repressor (FIR) expression or the altered splicing of FIR may contribute to the pathogenesis of type acute lymphocytic/ lymphoblastic leukemia (T-ALL) via upregulating c-MycNotch1 axis independent on TP53 (Figure 7E)
Figure7: Alternative splicing of FIR connects DNA damage response, c-myc activation and cell cycle control. (A) 20 and 50pmol of TP53 siRNA were transfected into HeLa cells

Summary

Introduction

DNA damage affects carcinogenesis, transcription, alternative splicing, and cell cycle control; the precise mechanism behind these affects remains largely unexplored. FUSE-binding protein (FBP) is a transcription factor that stimulates c-myc expression [1,2,3]. A splice variant of FIR that lacks exon 2 in the transcriptional repression domain (FIRΔexon2) elevates c-Myc protein expression in vitro [11]. FIRΔexon mRNA is frequently upregulated in human colorectal cancers [12] as well as hepatocellular carcinoma [13], where it stimulates tumor growth by preventing FIR from suppressing c-myc [13]. Recent studies suggested that DNA damage induces alternative splicing of several genes including FIR [14,15]. DNA damage may induce persistent c-myc upregulation via FIRΔexon in cancer cells, whereas it induces TP53 in normal cells

Methods

Results

Conclusion